The long term goals of this project are to elucidate the mechanism by which growth factors activate the amiloride-sensitive Na transport pathway in cultured human fibro-blasts and to assess the role of the growth factor-stimulated Na flux in regulating mitogenesis. The specific aims of this research proposal are directed principally attesting our hypothesis that growth factors regulate Na influx by elevating intracellular Ca activity which results in a calmodulin-dependent activation of the Na transport pathway. We propose to study the mobilization of intracellular Ca in response to treatments which activate Na influx and to investigate two possible mechanisms (phosphorylation of the transport protein and disruption of microtubules) for calmodulin regulation of Na flux. The mechanism for the rapid inactivation of the serums-stimulated Na influx pathway will be investigated to determine whether it results from growth factor-receptor down regulation or from negative feedback signals due to elevated intracellular Na concentration. We will determine whether lys-bradykinin, which we have demonstrated to be a potent stimulator of Na influx, also stimulates DNA synthesis in human fibroblasts and whether a correlation between Na influx and DNA synthesis stimulated by various combinations of peptides (EGF, vasopressin, lysbradykinin) can be demonstrated. We will investigate the involvement of protons in the Na influx process and in cell growth regulation. Attempts will be made to select Na transport mutants from Chinese Hamster Ovary (CHO) cells and to demonstrate an up-regulation of the Na transport pathway in human fibroblasts grown in low Na medium. Experiments designed to address these specific aims involve measurement of ion fluxes across cellular membranes, measurement of intracellular Ca activity using a fluorescence indicator, measurement of binding of growth factors to membrane receptors, measurement of DNA synthesis using 3H-Thd incorporation, and measurement of protein phosphorylation in cultured human fibroblasts. In addition, the "tritium suicide" technique will be used for selection of transport negative mutants in CHO cells.